Vertebral malformations (VMs) represent conditions that occur with an estimated incidence of 1/2000 and pose a significant public health impact due to their association with congenital scoliosis (CS) and other conditions. In order to better understand genetic variants that contribute to VM, we propose to perform whole exome sequence analysis (WES) in 86 patients with VM through the Baylor Hopkins Center for Mendelian Genetics and Victor Chang Cardiac Research Institute. Following rigorous bioinformatics analysis sequence variants have been identified so far in COL11A2. To determine the requirement for col11a2 in vertebral development, we will employ established CRISPR/Cas9 gene targeting approaches to generate zebrafish col11a2 loss-of-function (LOF) and VM-associated alleles. Caudal vertebral fusions have been observed in our laboratory in stable F2 col11a2 LOF mutants, which supports a role for col11a2 in vertebral development. To investigate the functional consequence and pathogenicity of COL11A2 R130W, R1407H, and R1413H VM-associated mutations, we will employ 2 parallel genetic strategies: (1)Create CRISPR/Cas9-based knock-in (KI) mutations; (2)Rescue LOF phenotypes with VM-associated col11a2 expression transgenes. The ability to identify genes associated with VM will serve as a foundation for classification of VM which can then be used identify novel pathways, novel phenotypes associated with patterning gene mutations, and to guide prevention and therapeutic strategies for patients cared for by the clinical genetics and greater pediatric orthopedic community.